A proof-of-concept study to investigate the efficacy of heat-inactivated autovaccines in Mycobacterium caprae experimentally challenged goats

This study aimed to assess the efficacy of a heat-inactivated Mycobacterium caprae (HIMC) vaccine in goats experimentally challenged with the same strain of M. caprae. Twenty-one goats were divided into three groups of seven: vaccinated with heat-inactivated Mycobacterium bovis (HIMB), with HIMC and unvaccinated. At 7 weeks post-vaccination all animals were endobronchially challenged with M. caprae. Blood samples were collected for immunological assays and clinical signs were recorded throughout the experiment. All goats were euthanized at 9 weeks post-challenge. Gross pathological examination, analysis of lung pathology using computed tomography, and bacterial load quantification in pulmonary lymph nodes (LN) by qPCR were carried out. Only HIMC vaccinated goats showed a significant reduction of lung lesions volume and mycobacterial DNA load in LN compared to unvaccinated controls. Both vaccinated groups showed also a significant reduction of the other pathological parameters, an improved clinical outcome and a higher proportion of IFN-γ-producing central memory T cells after vaccination. The results indicated that homologous vaccination of goats with HIMC induced enhanced protection against M. caprae challenge by reducing lung pathology and bacterial load compared to the heterologous vaccine (HIMB). Further large-scale trials are necessary to assess the efficacy of autovaccines under field conditions.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis complex bacteria that affects humans and a wide range of domestic animals and wildlife. Animal TB is a zoonotic disease that still represents an important public health issue, particularly in middle and low-income regions where it is estimated to cause up to 10% of human TB cases in some countries 1 . Hence, the control and eradication of TB requires a One Health approach.
Domestic goats are particularly susceptible to this disease 2 , which is mainly caused by Mycobacterium caprae and Mycobacterium bovis 3 . The goat population in Spain was approximately 2.7 million in 2020 FAOSTAT, accessed on 12/07/2022), being the second largest census of the European Union after Greece. Goat herds are not yet subjected to a National eradication program, except for those epidemiologically related with cattle 4

Results
Cell-mediated immune responses after vaccination and challenge. The mean antigen-specific whole blood IFN-γ responses before and after challenge for each treatment group are depicted in Fig. 1. A mild increase of IFN-γ responses to PPD-B and P22 were detected in the HIMC vaccinated group at seven weeks after vaccination (Week 0, Fig. 1a,b) although without significant difference when compared to other groups. After challenge, the mean levels of IFN-γ began to increase in the three experimental groups. At week 5, PPD-B specific IFN-γ responses were significantly higher in unvaccinated animals compared to HIMC and HIMB groups (P < 0.05 and P < 0.01, respectively Fig. 1a). At the same time point, both vaccinated groups also showed lower P22 specific IFN-γ responses compared to the unvaccinated group although they were only statistically significant in the HIMB vaccinated group (P < 0.01, Fig. 1b). None of the vaccinated animals showed detectable IFN-γ responses to E/C after vaccination (Fig. 1c). After challenge, the vaccinated groups also showed lower E/Cspecific IFN-γ responses than the control group, being statistically significant at weeks 3 (P < 0.05) and 5 (P > 0.05 and P < 0.01 in HIMC and HIMB groups, respectively).
Analysis of IFN-γ-producing cell subsets was performed by flow cytometry at seven weeks post-vaccination (Week 0) using PBMC isolated from the seven HIMB vaccinated, 5 HIMC vaccinated and 5 unvaccinated animals (4 animals did not show sufficient viable PMBC for the assay). Gating strategy is shown in Fig. 2a-d. Both vaccinated groups showed statistically significant higher frequencies of IFN-γ-producing CD4 lymphocytes with a central memory phenotype (C45RO + CD62L + ) compared to unvaccinated animals (HIMB: P < 0.01, HIMC: P < 0.05, Fig. 2e).
Finally, specific production of multiple cytokines after stimulation of whole blood with PPD-B and E/C was analysed at week 0 (before challenge) and at weeks 5 and 9 post-challenge. Results of cytokine production profiles relative to maximum value of each cytokine are presented in Fig. 3. Before challenge (week 0, 7 weeks post-vaccination), both vaccinated groups showed a slightly higher PPD-B-specific proinflammatory (IFN-γ, IL-17A and IL-6) responses compared to the control group (Fig. 3a). Productions of IL-1β in both PPD-B and E/C stimulated blood samples were higher in the HIMB group (P < 0.001).
At five weeks after challenge, unvaccinated animals experienced an increase of both proinflammatory cytokines (IFN-γ, IL-17A, IL-1β) and IL-10 production. However, overall cytokine responses decreased in the control group at week 9, when HIMC vaccinated goats showed higher E/C-specific levels of IL-6 compared to Response against E/C antigen cocktail. *P < 0.05, **P < 0.01, Kruskal Wallis test with post hoc Dunn's test. Groups: Control (n = 7, green), HIMB (n = 7, blue), HIMC (n = 7, red). Two animals of the control group were humanely sacrificed at week 7.  Fig. 3b), and HIMB group showed significantly higher levels of IL-1b to PPD-B (P < 0.05, Fig. 3a). At this time point, vaccinated groups also showed a mild increase of both E/C-specific IFN-γ and antiinflammatory responses (IL-10 and IL-4 in HIMC and HIMB groups, respectively), although not statistically significant (Fig. 3b). No statistically significant differences in TNFα production were observed among groups throughout the experiment.
Humoral responses after vaccination and challenge. IgG levels to the antigen MPB83 (Fig. 4a) and the P22 antigenic complex (Fig. 4b) were measured by two ELISAs throughout the study showing similar kinetics at 3 and 7 weeks postvaccination (p.v). HIMB-vaccinated group showed an increase of IgG levels to both antigens compared to HIMC and control groups (Fig. 4), although this increase was only statistically significant in the P22 ELISA (P < 0.05, Fig. 4b). Only two HIMC vaccinated animals showed mild IgG responses after vaccination. By contrast, the IgG responses to both antigens increased at 3 weeks post-challenge in both vaccinated groups compared to the control group, although it was only statistically significant in the P22 ELISA (P < 0.01 and P < 0.05, for HIMB and HIMC groups, respectively), that showed a slightly delayed seroconversion. From week 5 onwards, no significant differences between groups were observed (Fig. 4).
Clinical signs and body condition after challenge. Animals were observed for clinical signs of TB after M. caprae challenge. Results are summarized in Table 1. Mild clinical signs appeared in most non-vaccinated animals at week 3 and evolved to severe clinical signs in all animals at week 7 (two of them were humanely euthanized because end point criteria were reached). Only a few vaccinated animals showed mild clinical signs at week 3 (one of the HIMC and two of the HIMB groups). At the end of the experiment almost all animals  Rectal temperature and body weight were measured weekly after challenge (Fig. 5). The mean rectal temperature of the vaccinated groups was significantly lower than the control group from week 2 onwards (P < 0.05, Fig. 5a). No significant differences were observed between vaccinated groups. The mean body weight gain was significantly higher in vaccinated groups compared to the control group from week 3 onwards (P < 0.05, Fig. 5b). At the end of the experiment, the mean cumulative weight increase of the control group was 2.3 kg (− 0.9 to 5.4, 95% CI), significantly lower (P < 0.05) than the vaccinated groups (HIMB: 6.1 kg, 95% CI 5-7.7; HIMC: 7.0 kg, 95% CI 1. 6-12.4). Again, no statistically significant differences were observed between HIMB and HIMC groups throughout the experiment.
With regard to extrapulmonary TB, the control group was the only one with all animals showing extrapulmonary lesions, a proportion significantly higher to that found in vaccinated groups (4/7 each, P < 0.05). A summary of extrapulmonary lesions found in each treatment group and their location is shown in Table 2.
Cross-sectional analysis: biomarkers of vaccine efficacy. To assess immunological markers as predictors of vaccination outcome, the antigen-specific IFN-γ-producing CD4 central memory T (TCM) cell subsets (CD45RO + CD62L + ) obtained by flow cytometry in each vaccinated animal (N = 12) were correlated with clinical, pathological and bacteriological results of the same animal (Table 3). Results showed significant inverse correlations of TCM % with volume of lung lesions and lesion volume / lung volume ratio, whereas no correlation was observed with LN lesions and bacterial DNA load in LN. Regarding clinical signs, body weight increase after challenge (mean Kg at week 9 minus mean Kg at week 0) showed significant direct correlation with TCM % and mean rectal temperature obtained at the temperature peak time point (week 3) showed significant inverse correlation with TCM %.

Discussion
Inactivated mycobacterial vaccines can be adapted ad hoc to field conditions by generating autovaccines with the same strains circulating in the herd or in a specific outbreak area. This is the first proof-of-concept study to evaluate the efficacy of a heat-inactivated autovaccine against M. caprae infection in goats.
Nowadays, there is a certain degree of background regarding the inactivated vaccine candidate based on M. bovis (HIMB), that have been reported to protect goats against TB in laboratory and field conditions 18,20 , as well as cattle 21 , wild boar 22,23 , red deer 24 , badgers 25 and wild pigs 26 . HIMB represents an interesting alternative to live-attenuated BCG vaccine since the deployment logistics are simpler and the strain survival is not required 27 . In the present study, the gross pathology and bacteriological results, as well as the clinical follow-up www.nature.com/scientificreports/ after experimental challenge with M. caprae, ratified that MTBC-inactivated vaccines conferred protection when compared to unvaccinated animals. Moreover, the HIMC group, vaccinated with the M. caprae strain homologous to the challenge strain (autovaccine proof-of-concept), showed slightly better protection against pulmonary TB compared to the group vaccinated with the heterologous vaccine (HIMB), with lower volume of lung lesions and animals showing lower number of affected lung lobes. In addition, the HIMC vaccinated group presented significantly lower bacterial DNA load in pulmonary LN, even when considering that one of the animals vaccinated with HIMC showed a huge amount of bacterial load (over 10 6 CFU equivalents, very much higher than any unvaccinated goat). This animal also showed very high pathological extension compared to the rest of the group (being an outlier for all parameters). Intriguingly, this goat did not show detectable immune responses (neither to ELISA nor IGRA) after vaccination. Even though an unnoticed incorrect administration of the vaccine cannot be ruled out, this animal could have been immunocompromised at the vaccination time point, and thus would have not been properly immunized.
Interestingly, vaccinated groups not only did show a reduced volume of lung lesions and lung mineralization compared to the control group, but also showed lower volume ratio of mineralization within the lung lesions, suggesting that the lung pathology was in earlier/more contained developmental stages compared to unvaccinated  Table 2. Number of animals with extrapulmonary TB lesions and their location in each treatment group (N = 7). *P < 0.05, **P < 0.01, ***P < 0.001, Chi-square test.  www.nature.com/scientificreports/ animals according to criteria previously described for M. bovis infected cattle using histopathology 28 . This approach defined four granuloma developmental stages according to different parameters such as the degree of mineralization. Using these criteria, cattle vaccinated with BCG or BCG-prime / heterologous boost regimes also showed lower proportion of lung lesions in advanced developmental stages 29,30 . Vaccination also reduced the incidence and spread of extrapulmonary TB as previously described in BCG or HIMB vaccinated goats 18 . All unvaccinated animals showed TB lesions in the spleen and, in this control group there was also a higher number of animals with lesions in hepatic and mesenteric LN, indicative of a reduced lymphohematogenous dissemination of mycobacteria in both vaccinated groups.

Total extra-pulmonary lesions
Comparisons of clinical parameters, particularly the body weight increase and rectal temperature kinetics, were very consistent with post-mortem results. The body weight change and the rectal temperature at 3 to 5 weeks post-challenge are useful indicators of vaccination outcome as previously demonstrated in experimental trials conducted in goats and sheep 18,31,32 , and should be considered as valuable parameters in experiments to assess the efficacy of vaccines and treatments against TB.
Even though the administration of inactivated vaccines induced detectable ex vivo IFN-γ responses to PPD-B and P22 using IGRAs, in a number of animals, no responses were detected when using an E/C protein cocktail after vaccination and before challenge. This result agrees to previously reported in HIMB parenterally vaccinated goats 18 . In addition, the intensity of E/C-specific IFN-γ responses after challenge were equivalent to that induced by PPD-B and P22, indicating that this cocktail, initially developed for TB diagnosis and compatible with BCG vaccination, could be also suitable as a DIVA reagent for IGRA tests after administration of MTBC inactivated vaccine.
Unvaccinated animals showed higher IFN-γ levels measured by the IGRAs after challenge compared to vaccinated ones, reaching a peak response at five weeks post-challenge. This trend was confirmed by increased proinflammatory responses in unvaccinated animals measured by the multi-cytokine assays performed at 5 weeks post challenge. At this time point, unvaccinated group showed higher antigen-specific IFN-γ, IL-17A and IL-1β levels compared to vaccinated groups. This multi-cytokine proinflammatory pattern has also been described in buffalos infected with M. bovis 33 that showed increased proinflammatory cytokines and chemokines (IP-10, IFNγ, IL-6, IL-17A, MIP-1β, MIP-1α and MCP-1) in whole blood stimulated with the E/C whereas anti-inflammatory cytokines (IL-4 and IL-10) remained in basal levels. On the contrary, in the present experiment unvaccinated goats also showed increased IL-10 responses at 5 weeks post challenge. The anti-inflammatory effect induced by this increase of IL-10 levels in unvaccinated goats could be associated to the subsequent decrease of proinflammatory responses (to both IGRA and multi-cytokine assays at week 9 post vaccination), while vaccinated animals maintained moderate levels throughout the experiment.
In this study, the contained IFN-γ responses in vaccinated animals agreed with a more favourable disease outcome. However, whole blood ex vivo IFN-γ responses in vaccine trials should be interpreted with caution since previous studies reported that BCG vaccinated goats showed reduced 34 , similar 35 or even higher 18 antigenspecific IFN-γ responses after M. caprae challenge compared to unvaccinated controls.
Therefore, more accurate immunomarkers are required as predictors of vaccine efficacy and correlators of disease progression. The most useful immunological biomarkers to predict the vaccine efficacy are those that can be measured before the infection/exposure of animals to mycobacteria. With this purpose, as well as to estimate the duration of the vaccine-induced immunity, we assessed the proportion of antigen-specific IFN-γ-producing TCM subsets, based on the expression of CD45RO and CD62L receptors 36 .
Individual PPD-B-specific IFN-γ-producing TCM % correlated with favorable disease outcome parameters, namely, reduced lung lesions, higher body weight gain, and lower peak of rectal temperature, suggesting that this immunological parameter can be a useful biomarker to infer the vaccine-induced protective immunity. Similarly, proliferation of antigen-specific memory T-cells determined by cultured IFN-γ ELISPOT were also inversely associated to the pathology degree in vaccinated cattle and goats 35,37 .
On the other hand, the expansion of cells with a TCM phenotype elicited by vaccination is suggestive of long-term immunity as reported for mice and humans 36,38 . Nevertheless, the duration of the vaccine-induced immunity cannot be properly estimated in merely a seven-week period after vaccination and longer studies are required to confirm the results. In long-term trials, a wane of BCG-induced ex vivo IFN-γ response has been observed in goats from 24 weeks p.v. onwards 9 and, approximately one year after vaccination, the proportion of PPD-B-specific IFN-γ-producing T-memory cells (CD45RO+) was similar to that of unvaccinated goats, although it increased significantly when measured three weeks after revaccination 32 .
Finally, serological results confirm the ability of inactivated vaccines, particularly HIMB, to induce MTBCspecific antibody responses 18 and, as such, the specificity of the serology will be compromised as a TB diagnostic tool in vaccinated animals 39 . Surprisingly, the seroconversion was less frequent and less intense (when it occurred) after HIMC vaccination. A lower expression of MPB83 (also a major component of P22 reagent) or a lower exposure of this protein in the M. caprae cell surface compared to M. bovis might be explanatory factors of this different antibody-response patterns, although the underlaying mechanisms were not investigated and further comprehensive and focused studies should be carried out to confirm this hypothesis.
Given that autovaccines may have advantages, particularly from the regulatory point of view, and that heatinactivated mycobacterial vaccines are easy to produce, the results of this proof-of concept study encourage to conduct further autovaccine field trials in natural infection settings to validate this vaccination strategy that can contribute to control TB in goats in high-prevalent scenarios when test-and-cull is unfeasible.

Methods
Animals, vaccination schedule and experimental infection. Twenty-one female Pyrenean breed goats of approximately 2 months of age, from an officially TB-free herd located at the Catalan Pyrenees (Spain) were divided into 3 experimental groups of 7 animals each: Unvaccinated control group, group vaccinated with heat-inactivated M. bovis (HIMB) and group vaccinated with heat-inactivated M. caprae (HIMC). Inactivated vaccines were produced at NEIKER (Basque Country, Spain) and prepared as previously described 22 . Briefly, HIMB was prepared from a M. bovis field strain (SB0339,www. Mbovis. org) isolated from a naturally infected wild boar, while HIMC was produced by the same procedure using a M. caprae field strain (SB0157, www. Mbovis. org) isolated from a tuberculous sheep in Catalonia 40 . Both field strains were sub-cultured in Middlebrook 7H9 broth (BD Diagnostics, USA) enriched with Oleic acid-Albumin-Dextrose-Catalase (OADC) for 3 weeks approximately and subsequently washed, tittered and inactivated as previously described 18 . Inactivated mycobacterial suspensions were oil-adjuvanted with Montanide ISA 61 VG (Seppic, Paris, France) as indicated by the manufacturer. Final concentration of vaccines was approximately 5 × 10 7 inactivated CFU/ml. All animals were subcutaneously vaccinated (on the right scapula) with 1 ml of HIMB or HIMC.
Six weeks after vaccination, all experimental animals were transferred to the Biosafety Level-3 (BSL-3) facility of IRTA-CReSA (Catalonia, Spain) and were housed in two experimental boxes with 3-4-4 and 4-3-3 animals from Control, HIMB and HIMC groups in each box, respectively. Animals were fed with hay, alfalfa, mineral salt and maintained with water ad libitum throughout the experiment.
After a week of acclimatation, the animals were sedated with an intramuscular administration of acepromazine maleate (0.05 mg/kg) and butorphanol (0.2 mg/kg) and subsequently anesthetized with the intravenous administration of propophol (5 mg/kg) and midazolam (0.2 mg/kg). Afterwards, the animals were endonbronchially challenged as previously described 2 with 0.5 ml of an approximately 2 × 10 3 CFU/ml suspension of the same M. caprae strain used to prepare the HIMC vaccine.
After challenge, the animals were daily monitored for clinical signs. Rectal temperature and body weight were measured every week. Animals were bled at weeks -7 (vaccination time point), -3, 0 (challenge time point), 3, 5, 7 and 9 (end point) for immunological assays.
The experimental design and the experiments were conducted following the recommendations in ARRIVE guidelines (https:// arriv eguid elines. org/). Ex vivo IFN-γ release assay (IGRA). Whole blood samples were collected at time points mentioned above from the jugular vein using heparinized tubes and subsequently stimulated in 96-well cell culture plates (Eppendorf, Hamburg, Germany) with PPD-B, P22 (except at week 7) and E/C at a final concentration of 20 μg/ml. Phosphate-buffered saline was used as an unstimulated control. Samples were incubated at 37 ºC and 5% CO 2 for 18 ± 2 h. Plasma supernatants were collected after centrifugation at 18 g for 10 min and the released IFN-γ was measured by ELISA according to the ID.screen® Ruminant IFN-g kit (ID.vet, Grabels, France) instructions. ELISA plates were read as optical density obtained at 450 nm (OD 450nm ) using a spectrophotometer (Biotek Power Wave XS®, Agilent, dana Clara, USA). Antigen-specific IFN-γ responses were calculated as ΔOD 450nm of antigen-stimulated well minus OD 450nm of non-stimulated well (ΔOD 450nm ).

Ethics statement.
Multi-cytokine quantification assay. Cytokines in plasma supernatants from PPD-B and E/C stimulated and unstimulated whole blood samples were quantified at weeks 0, 5 and 9 post-challenge using a bovine customized multiplex assay kit (MILLIPLEX® Millipore, Merck Life Science S.L.U., Madrid, Spain) including a seven-plex panel for IL-1β, TNFα, IL-6, IL-10, IL-4, IFN-γ and IL-17A. 25 μl of each plasma sample and cytokine standards were analysed following the manufacturer's instructions using xMAP® technology (Thermofisher Scientific, Waltham, MA, USA). ELISA plates were read on MAGPIX instrumental platform with xPONENT acquisition software (Thermofisher Scientific).

Antibody detection assays. Plasma samples from all experimental animals were analysed in duplicate to
follow-up the antibody responses to MTBC after vaccination and challenge. Two indirect ELISAs were used to detect total IgG against the MPB83 antigen and the P22 antigenic complex, respectively. The two ELISAs were performed as previously described 42,43 . MPB83-IgG levels were calculated as OD450nm of antigen-coated well minus OD450nm of non-coated well (ΔOD450nm), whereas P22-IgG levels were calculated as ELISA percentage (E%) = [mean OD450nm of antigen-coated well/(2 × mean negative control OD450nm)] × 100.
Necropsy, pathological examination and computed tomography (CT). Experimental animals were euthanized at 9 weeks post-challenge with an overdose of intravenous sodium pentobarbital (two goats from the control group were humanely euthanized at week 7). At necropsy, the diameter of TB compatible lesions in retropharyngeal (right and left), mediastinal (cranial and caudal) and tracheobronchial lymph nodes (LN) were measured, and the volume of lesions was inferred as previously described 31 . Other visible lesions in extrapulmonary tissues were also recorded and fixed in 10%-buffered formalin to be confirmed by histopathology and the Ziehl Neelsen stain to detect acid-fast bacilli. Lungs were also formalin-fixed, including airways perfusion with formalin before immersion, as previously described 2 , and then scanned by computed tomography (CT) using a 16-slice multi-detector scanner (Brivo CT-385, General Electric Healthcare, Madrid, Spain). CT analysis was performed as previously described 31 . Briefly, total volumes of lungs and TB lesions were measured using volume rendering images with different density patterns (i.e. solid, mineralized and cavitary lesions), and total volume of mineralization was calculated using 100-300 Hounsfield units.
Bacterial DNA quantification. After macroscopic pathologic evaluation, the whole mediastinal and tracheobronchial LNs were stored at − 20 °C until they were processed for bacteriology (one month later). Then LNs were thawed and homogenized in 10 ml of sterile distilled water using a homogenizer (Masticator, IUL Instruments, Barcelona, Spain). An aliquot of 1 ml of each homogenate was inactivated at 75 °C for 1 h and, in parallel, an aliquot of 100 ml of the M. caprae master seed (~ 10 8 CFU/ml), used for the challenge inoculum, was also inactivated and then ten-fold serially diluted to establish the standard curve. DNA samples were extracted using the ID Gene™ spin universal extraction kit (ID.vet) and amplified with the Mycobacterium tuberculosis complex Duplex kit (ID.vet). Amplification was performed in a 7500 fast real-time PCR system (Applied Biosystems, Walham, MA, USA). CFU genomic equivalents were calculated as previously described 44 .
Data analysis. A completely randomized design was carried out for studying the effects of vaccinations as a primary factor. Temperature and body weight were compared by using unidirectional ANOVA followed by Tukey test for multiple comparisons. Antigen-specific immune responses (measured by IGRA, IgG-ELISA, Multi-cytokine assay and Flow Cytometry) were compared by using Kruskal-Wallis test with post hoc Dunn's test. Volumes of lesions in LN and lungs (measured by direct visual inspection and CT, respectively), and mycobacterial DNA load (measured by qPCR) were compared by using Kruskal-Wallis test with post hoc one-tailed Dunn's test.
Correlations of flow cytometry results (% of antigen-specific IFN-γ-producing T-memory cell subsets) with clinical and post-mortem parameters were assessed by using one-tailed non-parametric Spearman's Rank correlation coefficient. A p-value less than 0.05 was considered statistically significant.
GraphPad Prism version 8.0.0 (San Diego, CA, USA) was used for the statistical analysis.

Data availability
The databases generated and analyzed during the current study are available from the corresponding author on a reasonable request.